The present invention provides lanthanide binding tags (LBT) that selectively complex trivalent lanthanide (Ln) ions and afford stable complexes with desirable physical properties, including at least one of fluorescence and X-ray scattering and anomalous X-ray scattering. These LBTs are useful as probes for applications in analytical biochemistry, biophysics, biotechnology, medicine, and proteomics.
One notable use is in the arena of determining protein structures. With the release of the sequence of the human genome, determining the structure of proteins of the genome-wide scale is a formidable task. Traditionally, each target gene is cloned into an expression vector, expressed with the use of a single set of conditions, and the resulting protein in then purified. With this protein in hand, a number of basic screens for crystallization are used, followed by further screening, if necessary, to optimize crystal quality. Lastly, the best crystal(s) are used for diffraction analysis.
Using conventional methods, the throughput of macromolecular 3D structure determination can be improved only by increasing the person-hours of work. As a consequence, academic and industry-based researchers have initiated research and development programs to develop high-throughput (HT) structure determination process pipelines. HT structural biology requires development of methods and reagents to streamline and automate the process of protein structure determination.
The early steps in the pipeline can capitalize on the HT technologies developed during genomic sequencing efforts. Robotic liquid handling and colony picking procedures, as well as automated sequencing, can easily be adapted for the cloning and expression steps. As these impediments are removed, other processes become rate limiting. For example, once sufficient quantities of purified protein are obtained, crystals suitable for x-ray diffraction must be grown and phase information obtained from these crystals in order to determine the structure. Lastly, the diffraction data collection phase determination, and analysis steps must be automated, streamlined, and made user-independent.
Along with other applications described herein, the LBTs of the present invention can be easily integrated into the covalent architecture of the protein products of the genes of interest and used to facilitate aspects of structure and function determining processes, including x-ray diffraction structure determination.